Figure 2: Genetic characterization of intragenic suppressors of TIF5 allele SUI5/G31R. (A) Derivatives of his4-301 tif5Δ strain ASY100 harboring a TIF5+URA3 plasmid and the indicated TIF5-FL alleles on LEU2 plasmids (hc WT on pAS5-132, sc WT on pAS5-101, G31R on pAS5-111, G31R,M18V on pAS5-115, G31R,K33E on pAS5-117, G31R,G62S on pAS5-112, G31R,L61A on pAS5-116 and hc G31R,T34N on pAS5-136) were replica-plated to SC-LU supplemented with either 0.3 mM histidine (-LU) or 0.0003 mM histidine (-LUH), or to SC-L supplemented with 5.2 mM 5-FOA (5-FOA). Cells were incubated for 3d (-LU) or 6d (-LUH and 5-FOA) at 30°C. (B) 10-fold serial dilutions of strains described in (A) were spotted on SC-LU and incubated for 3d at 30°C. (C) Derivatives of tif5Δ strain ASY137 harboring a TIF5+TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with HIS4-lacZ reporter plasmids with AUG (p367) or UUG (p391) start codons. Cells were cultured in SC lacking leucine, tryptophan and uracil at 30°C and β-galactosidase activities were measured in whole cell extracts (WCEs). Ratios of β-galactosidase expressed from the UUG to AUG reporter were calculated from three independent transformants and mean ratios and and S.E.M.s (error bars) were plotted. (D) Slg− and His+/Sui− phenotypes of the his4-301 tif5Δ strains harboring TIF5 alleles described in (A), and isogenic strains containing M18V on pAS5-106, K33E on pAS5-108, G62S on pAS5-103, L61A on pAS5-107 or hc T34N on pAS5-135, were determined by spotting serial 10-fold dilutions on SC-LU supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (E) Derivatives of his4-301 sui1Δ strain ASY250 with the chromosomal TIF5 gene under the GAL1 promoter (PGAL-TIF5) harboring SUI1+ on a TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with the AUG or UUG HIS4-lacZ reporter plasmids. Transformants were cultured in synthetic minimal medium with 2% galactose as carbon source and supplemented with 0.3 mM histidine (SGal+H) and then shifted to synthetic minimal medium with 2% glucose (SD+H) for 16 h. UUG:AUG initiation ratios for the HIS4-lacZ reporters were determined as in (C). (F) HIS4-lacZ UUG:AUG initiation ratios were measured as in (C) for strains described in (D) harboring the indicated plasmid-borne TIF5 alleles. For panels A and D, images have been cropped from results obtained from different plates examined in parallel in the same experiments.

Mentions:
M18V strongly suppresses the dominant His+/Sui− phenotype, but not the recessive lethality, conferred by G31R. Thus, transformants harboring tif5-G31R,M18V fail to grow on −His medium in the presence of TIF5+, indicating the Ssu− phenotype, and also cannot grow on 5-FOA medium, indicating inviability of the cells following eviction of TIF5+ (Figure 2A, rows 3–4). The failure to grow on the −His medium does not result from a general growth defect, as the tif5-G31R,M18V strain grows better on +His medium than does the parental His+tif5-G31R strain, which displays the dominant Slg− phenotype conferred by G31R (Figure 2B, rows 3–4 versus row 2).

Figure 2: Genetic characterization of intragenic suppressors of TIF5 allele SUI5/G31R. (A) Derivatives of his4-301 tif5Δ strain ASY100 harboring a TIF5+URA3 plasmid and the indicated TIF5-FL alleles on LEU2 plasmids (hc WT on pAS5-132, sc WT on pAS5-101, G31R on pAS5-111, G31R,M18V on pAS5-115, G31R,K33E on pAS5-117, G31R,G62S on pAS5-112, G31R,L61A on pAS5-116 and hc G31R,T34N on pAS5-136) were replica-plated to SC-LU supplemented with either 0.3 mM histidine (-LU) or 0.0003 mM histidine (-LUH), or to SC-L supplemented with 5.2 mM 5-FOA (5-FOA). Cells were incubated for 3d (-LU) or 6d (-LUH and 5-FOA) at 30°C. (B) 10-fold serial dilutions of strains described in (A) were spotted on SC-LU and incubated for 3d at 30°C. (C) Derivatives of tif5Δ strain ASY137 harboring a TIF5+TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with HIS4-lacZ reporter plasmids with AUG (p367) or UUG (p391) start codons. Cells were cultured in SC lacking leucine, tryptophan and uracil at 30°C and β-galactosidase activities were measured in whole cell extracts (WCEs). Ratios of β-galactosidase expressed from the UUG to AUG reporter were calculated from three independent transformants and mean ratios and and S.E.M.s (error bars) were plotted. (D) Slg− and His+/Sui− phenotypes of the his4-301 tif5Δ strains harboring TIF5 alleles described in (A), and isogenic strains containing M18V on pAS5-106, K33E on pAS5-108, G62S on pAS5-103, L61A on pAS5-107 or hc T34N on pAS5-135, were determined by spotting serial 10-fold dilutions on SC-LU supplemented with 0.3 mM His (+His) or 0.0003 mM His (−His) and incubated for 3d (−His) or 6d (+His) at 30°C. (E) Derivatives of his4-301 sui1Δ strain ASY250 with the chromosomal TIF5 gene under the GAL1 promoter (PGAL-TIF5) harboring SUI1+ on a TRP1 plasmid and the indicated TIF5 alleles on LEU2 plasmids were transformed with the AUG or UUG HIS4-lacZ reporter plasmids. Transformants were cultured in synthetic minimal medium with 2% galactose as carbon source and supplemented with 0.3 mM histidine (SGal+H) and then shifted to synthetic minimal medium with 2% glucose (SD+H) for 16 h. UUG:AUG initiation ratios for the HIS4-lacZ reporters were determined as in (C). (F) HIS4-lacZ UUG:AUG initiation ratios were measured as in (C) for strains described in (D) harboring the indicated plasmid-borne TIF5 alleles. For panels A and D, images have been cropped from results obtained from different plates examined in parallel in the same experiments.

Mentions:
M18V strongly suppresses the dominant His+/Sui− phenotype, but not the recessive lethality, conferred by G31R. Thus, transformants harboring tif5-G31R,M18V fail to grow on −His medium in the presence of TIF5+, indicating the Ssu− phenotype, and also cannot grow on 5-FOA medium, indicating inviability of the cells following eviction of TIF5+ (Figure 2A, rows 3–4). The failure to grow on the −His medium does not result from a general growth defect, as the tif5-G31R,M18V strain grows better on +His medium than does the parental His+tif5-G31R strain, which displays the dominant Slg− phenotype conferred by G31R (Figure 2B, rows 3–4 versus row 2).